Fig. 4

RC48 and dabrafenib synergistically inhibited the antitumor activity of A2058 and SK-MEL-28 cells in vitro. A Cytotoxicity of a combination with RC48 and dabrafenib (DAB) in A2058 and SK-MEL-28 cells as determined by CellTiter-Glo® Assay in accordance with the manufacturer’s instructions. CDI < 1 represents a synergistic effect. B A2058 and SK-MEL-28 cells were incubated with 2 μg/mL RC48, 1 μM DAB, or RC48 + DAB for various times. The cell confluency (%) was calculated using Incucyte S3 Zoom software based on phase contrast images from 0 h to 72 h. C The cell confluency (%) was shown for RC48 and DAB alone or in combination at 72 h. D A2058 and SK-MEL-28 cells were incubated with 2 μg/mL RC48, 1 μM DAB, or their combination for 24 h. Then A2058 cells were stained with Azide 555 (red) to detect EdU and DAPI (blue). Fluorescence images were obtained and analyzed with a confocal laser scanning microscope. Images captured at 200× magnification. Scale bars, 50 µm. E A2058 and SK-MEL-28 cells were treated with different treatments for 48 h. The cells were stained with an anti-Annexin V-FITC antibody and PI for apoptosis analysis using flow cytometry. F A2058 and SK-MEL-28 cells were treated with different treatments for 48 h. Western blotting was used to examine the expression levels of PARP and Mcl-1. GAPDH was used as a loading control. G A2058 and SK-MEL-28 cells were exposed to different treatments for 24 h. Cell motility was detected using Transwell migration and invasion assays. H A2058 and SK-MEL-28 cells were exposed to different treatments of 2 μg/mL RC48, 1 μM DAB, or RC48 + DAB for 24 h. Western blotting was used to examine the expression of EMT markers (SNAI1, N-cadherin, ZEB1). GAPDH was used as a loading control. Data represent the mean ± SEM of at least three independent experiments, and statistical significance was assessed using an unpaired t-test; *p < 0.05, **p < 0.01, ***p < 0.001 compared with control groups